Mammalian cells that are incapable of proliferating in suspended liquid culture but can be made to proliferate on the surface of a substrate are said to be anchorage-dependent.
Epithelial cells, such as keratinocytes, are anchorage dependent. Such cells cultured in the presence or a substrate which is non-inhibitory and non-cytotoxic will multiply in stratified colonies and eventually produce a confluent layer. Cell cultures of this type are used to investigate skin growth and have been used as skin crafts. Various technical capers have been published which describe in vitro techniques for growing skin cells and their subsequent use in the treatment of full-thickness wounds. For example E. Bell et al (J Invest Derm 81; 2s-10s 1983); E. Bell et al (Science 211; 1052-1054 1981); D. Asselineau and M. Pruneiras (Br J Derm 1984 III, Supplement 27, 219-222) and J. F. Burke et al (Ann Sura 94; 413-428 1981).
The ability of cells to anchor go a particular substrate is dependent on the properties of the substrate itself as well as the culturing conditions and the components of the culture medium. Culturing is usually carried out in hard plastic flasks made from a material which is substantially inert to the growth media and is non-cytotoxic to the cells. Polystyrene is a commonly used material for culture flasks.
One of the problems in using hard plastic flasks for the culture of epithelial cells for use as skin grafts is that the sheet of cells normally has to reach confluence before they can be harvested. The time taken to reach confluence may be long. Furthermore the layer of cells is not very strong mechanically and can easily be damaged when the unsupported cells are dislodged and handled unsupported.
It is known that the sheet of cells can be supported after it has been dislodged from the surface in order to facilitate handling and transfer to the wound surface. However this technique does not overcome the problems associated with hard surface cultures or the risks associated with dislodgement of the cells.
Epithelial cells have also been grown on natural materials such as collagen which can then be used directly as, a skin replacement. The techniques are described in the papers referred to hereinbefore. whilst epithelial cells grow well on collagen there are several disadvantages in using such a material. Since collagen is a natural substance it is not well defined and can vary substantially from one batch to another which is clinically undesirable if it is to be used as a skin graft. Collagen is also a difficuilt material to work with in the laboratory and it is a complex and time consuming process to isolate it, making it expensive to produce. A further disadvantage is that because collagen is a protein it cannot be easily sterilised by methods such as steam penetration as it will be denatured. Accordingly it is difficult to store and to keep sterile.
In an attempt to overcome the problems associated with the use of collagen and hard plastic surfaces as cell culture substrates it has been proposed to grow cells on the surface of water-swellable hydrophilic synthetic polymers. The cells are not immersed in the aqueous medium but are grown at the substrate-gaseous interface. The resultant sheet is celatively bulky making it poorly conformable and the cell layer has to be fully confluent before it can be transferred to the wound site which can take a considerable time for example 14-21 days. Such a process is described in PCT Publication No. W088/08448.